Parkinson's disease is a common age-related neurodegenerative disease characterized pathologically by a loss of dopaminergic neurons in the substantia nigra with resultant depletion of striatal dopamine and presence of Lewy bodies in the remaining neurons. Lewy body contains numerous functional and structural proteins, including alpha-synuclein and ubiquitin, and aggregation of alpha-synuclein is thought to be important in Lewy body formation as well as neurodegeneration, although the detailed mechanisms remain to be defined. Increasing evidence has suggested that mitochondrial dysfunction, increased oxidative stress and dysfunction of the ubiquitin-proteasome system may be involved in Lewy body formation as well as neurodegeneration. A few neurotoxicants, e.g. rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine, all capable of inhibiting mitochondria and enhancing oxidative stress, have been utilized widely to generate parkinsonian models in vitro and in vivo. Interestingly only rotenone produces Lewy body-like cytoplasmic inclusions. On the other hand, there are also human nigral degenerative diseases with or without Lewy body formation in the substantia nigra, including Parkinson's disease, dementia with Lewy body disease and multiple system atrophy. Our preliminary studies in cell cultures demonstrated that proteins interacting with alpha-synuclein may influence alpha-synuclein aggregation, Lewy body formation and cell death. Hence, this application proposes to advance our understating of the development of Lewy bodies as well as molecular mechanisms of development of Parkinson's disease with subtractive proteomics by looking for differences in the proteins associated with alpha-synuclein in the substantia nigra between rotenone-exposed and control rats first, and then comparing the protein profiles in rotenone-exposed rats vs. rats exposed to 1- methyl-4-phenylpyridinium, the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, or 6- hydroxydopamine. A second phase would examine tissues from patients with Parkinson's disease vs. controls as well as patients with multiple system atrophy. Finally, we will study the roles of the common and differentially displayed proteins, those related to oxidative stress, mitochondrial and proteosomal function in particular, in alpha-synuclein aggregation, Lewy body formation and neurodegeneration.